The long-term goal of the proposed research is to understand how a family of G protein-coupled receptors (GPCRs), called adhesion GPCRs, transduces signals. Adhesion GPCRs have been recognized in recent years as critical regulators of diverse biological processes, but the mechanisms of their regulation remain unclear. A unique feature of adhesion GPCRs is that they are typically cleaved into two fragments. How these fragments contribute to their functions is not known. Substantial preliminary data have showed that the two cleaved fragments (GPRN and GPRC) of GPR56, a member of adhesion GPCRs, interact to regulate downstream signaling events, including the secretion of the vascular endothelial growth factor (VEGF) and the activation of protein kinase C (PKC) in melanoma cells. The goal of the proposed research is to use this system to further dissect the activation mechanisms of GPR56 and its signaling components. Three specific aims are proposed: 1) Investigate whether GPRN regulates activities of GPRC. ELISA, co-immunoprecipitation, cell surface labeling, and mutagenesis analyses will be performed. 2) Examine whether GPRC regulates PKC activity through G proteins. Minigene construct expression, small GTPase activity assay, reporter assays, and PKC membrane translocation analyses will be performed. 3) Investigate the desensitization mechanisms of GPRC. Mutagenesis analyses, radiolabeling assay, and expression analyses of shRNAs and mutant arrestins will be performed. Taken together; the outcome of the research will help to establish a paradigm on the signal transduction of adhesion GPCRs, a group of receptors that play essential roles in human development and diseases. Thus the proposed research is highly relevant to the mission of the National Institute of General Medical Sciences of NIH.